PROJECT SUMMARY Infectious stocks of SARS-CoV-2 are now generally studied under BSL3 containment limiting the number of researchers who can work with it and increasing the difficulties of performing some of their experi- ments. We shall develop a derivative of SARS-CoV-2 that is replication-competent, but propagation- defective, supporting only a single round of infection, and therefore safe to examine under BSL2 contain- ment. Many more scientists will then be able to study this pathogen. This derivative, termed CoV-2.def, will be carried in cells such that its expression is repressed and can only be transcribed upon treatment with an inducer, doxycycline. It will also have two deletions of the structural proteins encoded by genes, E and M, so it will not be infectious in their absence. Neither of these engineered viral genes will have homology to CoV-2.def thus minimizing the chance of their recombining with CoV-2.def. M will be supplied in trans and can be expressed only upon induction. E will be supplied only by transfection of either an mRNA or the protein itself. These latter properties are designed to ensure that the cells that carry CoV-2.def do not accumulate viral RNAs during their passage that could contribute to recombina- tion and that the derivative can infect cells for only a single round. The derivative CoV-2.def will be constructed in multiple phases in order to ensure its safety and function- ing at each step. The first two orfs, 1A and 1B, which encode non-structural proteins of SARS-CoV-2, will be introduced into a plasmid vector derived from an Epstein-Barr Viral plasmid replicon. These orfs comprise the first 2/3 of the viral genome and will be regulated by the binding of a Tet-KRAB repressor so that they can be expressed only following induction by treatment with doxycycline. This construction will be examined for its conditional expression and for its dependence on M and E and perhaps on the N gene too for its release in extracellular particles. Only when these properties are established as being effective and safe will the intact CoV-2.def be constructed and tested under BSL3 containment. After CoV-2.def is found to be replication-competent, propagation-defective, and support only a single round of infection, it can be examined safely in BSL2 labs. Two sets of experiments with CoV-2.def will be conducted to improve treatment of patients with COVID- 19. Because CoV-2.def supports one round of infection, it can and will be used to measure titers of neutralizing antibodies in the plasma of patients. Neutralizing antibodies can only be measured with infectivity assays so that CoV-2.def is a powerful, safe tool with which to evaluate this facet of the adap- tive immune response and correlate it with patient outcomes. We shall measure these titers in samples provided by the Translational Science BioCore (TSB) BioBank, which is a shared service at the Univer- sity of Wisconsin Carbone Cancer Center, and be able to assess how being a cancer patient may affect this immune response to COVID-19. It is also clear that an effective, safe assay for the titers of neutraliz- ing antibodies can be used to identify samples of plasma that can be provided therapeutically to patients with COVID-19. In the second set of experiments, engineered derivatives of CoV-2.def will be used in two complementary CRISPR/Cas9 screens to identify cell-dependencies of SARS-CoV-2. We shall identify these cellular dependencies by establishing a library of gene knockouts with CRISPR/Cas9, infecting this library with two engineered derivatives of CoV-2.def to select for and against the cells that support infection, and determining the responsible genes by sequencing the sgRNAs in the selected populations. Inactivating these genes in our confirmatory experiments should block infection by CoV-2.def and thereby highlight cellular genes and pathways which are targets for anti-viral therapies.